Application of mRNA for use as a therapeutic against tumour diseases

ABSTRACT

The present invention relates to a pharmaceutical composition comprising at least one mRNA comprising at least one coding region for at least one antigen from a tumor, in combination with an aqueous solvent and preferably a cytokine, e.g. GM-CSF, and a process for the preparation of the pharmaceutical composition. The pharmaceutical composition according to the invention is used in particular for therapy and/or prophylaxis against cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/840,305, filed Aug. 31, 2015, which is a continuation of U.S.application Ser. No. 14/325,850, filed Jul. 8, 2014, now U.S. Pat. No.9,155,788, which is a divisional of U.S. application Ser. No.13/106,548, filed May 12, 2011, now abandoned, which is a divisional ofU.S. application Ser. No. 10/870,110, filed Jun. 18, 2004, now U.S. Pat.No. 8,217,016, which is a continuation under 35 U.S.C. §111(a) ofInternational Application No. PCT/EP02/14577, filed Dec. 19, 2002, whichclaims priority under 35 U.S.C. §119 to German Patent Application No.:101 62 480.8, filed Dec. 19, 2001. The entire text of each of theabove-referenced disclosures is specifically incorporated herein byreference without disclaimer.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted intext format via EFS-Web and is hereby incorporated by reference in itsentirety. Said text file, created Dec. 9, 2015, is namedCRVCP0009USC6.txt and is ˜2 KB in size.

BACKGROUND OF THE INVENTION

The present invention relates to a pharmaceutical composition comprisingat least one mRNA comprising at least one coding region for at least oneantigen from a tumour, in combination with an aqueous solvent andpreferably a cytokine, e.g. GM-CSF, and a process for the preparation ofthe pharmaceutical composition. The pharmaceutical composition accordingto the invention is used in particular for therapy and/or prophylaxisagainst cancer.

Gene therapy and genetic vaccination are molecular medicine methodswhich, when used in the therapy and prevention of diseases, will haveconsiderable effects on medical practice. Both methods are based on theintroduction of nucleic acids into cells or into tissues of the patientand on subsequent processing of the information coded by the nucleicacids introduced, i.e. expression of the desired polypeptides.

The conventional procedure of methods of gene therapy and of geneticvaccination to date is the use of DNA to insert the required geneticinformation into the cell. Various methods for introducing DNA intocells have been developed in this connection, such as e.g. calciumphosphate transfection, polyprene transfection, protoplast fusion,electroporation, microinjection and lipofection, whereas lipofection inparticular having emerged as a suitable method.

A further method which has been proposed in particular in the case ofgenetic vaccination methods is the use of DNA viruses as DNA vehicles.Such viruses have the advantage that because of their infectiousproperties a very high transfection rate can be achieved. The virusesused are genetically modified, so that no functional infectiousparticles are formed in the transfected cell. In spite of this safetyprecaution, however, a certain risk of uncontrolled propagation of thegenes having a gene therapy action and the viral genes introduced cannotbe ruled out because of possible recombination events.

The DNA introduced into the cell is conventionally integrated into thegenome of the transfected cell to a certain extent. On the one hand thisphenomenon can exert a desired effect, since a long-lasting action ofthe DNA introduced can thereby be achieved. On the other hand, theintegration into the genome results in a substantial risk of genetherapy. Thus e.g. the DNA introduced may be inserted into an intactgene, which represents a mutation which interferes or even completelyswitches off the function of the endogenous gene. On the one hand enzymesystems which are essential for the cell may be switched off by suchintegration events, and on the other hand there is also the danger of atransformation of the cell modified in this way into a degenerated stateif a gene which is decisive for regulation of cell growth is modified bythe integration of the foreign DNA. A risk of the development of cancertherefore cannot be ruled out when using DNA viruses as genetherapeutics and vaccines. In this connection it is also to be notedthat for effective expression of the genes introduced into the cell, thecorresponding DNA vehicles contain a strong promoter, e.g. the viral CMVpromoter. Integration of such promoters into the genome of the treatedcell can lead to undesirable changes in the regulation of geneexpression in the cell.

A further disadvantage of the use of DNA as gene therapeutics andvaccines is the induction of pathogenic anti-DNA antibodies in thepatient, causing a possibly fatal immune response.

In contrast to DNA, the use of RNA as a gene therapeutic or vaccine isto be classified as substantially safer. In particular, RNA does notinvolve the risk of being integrated into the genome of the transfectedcell in a stable manner. Furthermore, no viral sequences, such aspromoters, are necessary for effective transcription. Moreover, RNA isdegraded considerably more easily in vivo. Apparently because of therelatively short half-life of RNA in the blood circulation compared withDNA, no anti-RNA antibodies have been detected to date. RNA cantherefore be regarded as the molecule of choice for molecular medicinetherapy methods.

Nevertheless, medical methods based on RNA expression systems stillrequire a solution to some fundamental problems before they are usedmore widely. One of the problems of using RNA is reliable cell- ortissue-specific efficient transfer of the nucleic acid. Since RNAusually proves to be very unstable in solution, it has not hitherto beenpossible, or has been possible only in a very inefficient manner, to useRNA as a therapeutic or vaccine by the conventional methods which areused with DNA.

RNA-degrading enzymes, so-called RNAases (ribonucleases), areresponsible for the instability. Even the smallest impurities ofribonucleases are sufficient to degrade RNA in solution completely. Thenatural degradation of mRNA in the cytoplasm of cells is very finelyregulated. Several mechanisms are known in this respect. Thus, theterminal structure is of decisive importance for a functional mRNA. Atthe 5′-end is the so-called “cap structure” (a modified guanosinenucleotide), and at the 3′-end a sequence of up to 200 adenosinenucleotides (the so-called poly-A tail). The RNA is recognized as mRNAand the degradation is regulated via these structures. Moreover, thereare further processes which stabilize or destabilize RNA. Many of theseprocesses are still unknown, but an interaction between the RNA andproteins often appears to be decisive for this. For example, an “mRNAsurveillance system” has recently been described (Hellerin and Parker,Annu. Rev. Genet. 1999, 33: 229 to 260), in which incomplete or nonsensemRNA is recognized by certain feedback protein interactions in thecytosol and is rendered accessible to degradation, the majority of theseprocesses being performed by exonucleases.

Some measures for increasing the stability of RNA and thereby renderingpossible its use as a gene therapeutic or RNA vaccine have been proposedin the prior art.

To solve the abovementioned problems of the instability of RNA ex vivo,EP-A-1083232 proposes a process for introduction of RNA, in particularmRNA, into cells and organisms, in which the RNA is in the form of acomplex with a cationic peptide or protein.

WO 99/14346 describes further processes for stabilizing mRNA. Inparticular, modifications of the mRNA which stabilize the mRNA speciesagainst the degradation by RNases are proposed. Such modificationsconcern on the one hand stabilization by sequence modifications, inparticular reduction of the C and/or U content by base elimination orbase substitution. On the other hand, chemical modifications, inparticular the use of nucleotide analogues, and 5′- and 3′-blockinggroups, an increased length of the poly-A tail and complexing of themRNA with stabilizing agents and combinations of the measures mentioned,are proposed.

The U.S. Pat. No. 5,580,859 and U.S. Pat. No. 6,214,804 disclose, interalia, mRNA vaccines and therapeutics in the context of “transient genetherapy” (TGT). Various measures for increasing the translationefficiency and the mRNA stability based above all on untranslatedsequence regions are described.

Bieler and Wagner (in: Schleef (ed.), Plasmids for Therapy andVaccination, chapter 9, pages 147 to 168, Wiley-VCH, Weinheim, 2001)report on the use of synthetic genes in connection with gene therapymethods using DNA vaccines and lentiviral vectors. The construction of asynthetic gag gene derived from HIV-1, in which the codons were modified(alternative codon usage) compared with the wild-type sequence such thatthey corresponded to the use of codons which are to be found in highlyexpressed mammalian genes, is described. By this means, the A/T contentin particular was reduced compared with the wild-type sequence. Theauthors find in particular an increased expression rate of the syntheticgag gene in transfected cells. Furthermore, in mice an increasedformation of antibodies against the gag protein was observed in miceimmunized with the synthetic DNA construct, and also an increasedcytokine release in vitro in transfected spleen cells of mice. Finally,an induction of a cytotoxic immune response was to be found in miceimmunized with the gag expression plasmid. The authors of this articleattribute the improved properties of their DNA vaccine substantially toa change, caused by the optimized codon usage, to the nucleo-cytoplasmictransporation of the mRNA expressed by the DNA vaccine. In contrast, theauthors consider the effect of the modified codon usage on thetranslation efficiency to be low.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is therefore based on the object of providing anew system for gene therapy and genetic vaccination for tumours whichovercomes the disadvantages associated with the properties of DNAtherapeutics and vaccines.

This object is solved by the embodiments of the present inventioncharacterized in the claims.

In particular, a pharmaceutical composition comprising at least one mRNAcomprising at least one coding region for at least one antigen from atumour, in combination with an aqueous solvent, is provided.

According to the invention, the expression “antigen from a tumour” meansthat the corresponding antigen is expressed in cells associated with atumour. According to the invention, antigens from tumours are thereforein particular those which are produced in the degenerated cellsthemselves. These are preferably antigens located on the surface of thecells. Furthermore, however, antigens from tumours are also those whichare expressed in cells which are (were) not themselves (or originallythemselves) degenerated but are associated with the tumour in question.These also include e.g. antigens which are connected withtumour-supplying vessels or (re)formation thereof, in particular thoseantigens which are associated with neovascularization or angiogenesis,e.g. growth factors, such as VEGF, bFGF etc. Such antigens connectedwith a tumour furthermore also include those from cells of the tissueembedding the tumour. Corresponding antigens of connective tissue cells,e.g. antigens of the extracellular matrix, are to be mentioned here.

According to the invention, in the pharmaceutical composition one (ormore) mRNAs is used for therapy or inoculation, i.e. vaccination, fortreatment or prevention (prophylaxis) of cancer diseases. Thevaccination is based on the introduction of an antigen (or severalantigens) of a tumour, in the present case the genetic information forthe antigen in the form of the mRNA which codes for the antigen(s), intothe organism, in particular into the cell. The mRNA contained in thepharmaceutical composition is translated into the (tumour) antigen, i.e.the polypeptide or antigenic peptide coded by the modified mRNA isexpressed, as a result of which an immune response directed against thispolypeptide or antigenic polypeptide is stimulated. In the present caseof the use as genetic vaccines for treatment of cancer, the immuneresponse is therefore achieved by introduction of the geneticinformation for antigens from a tumour, in particular proteins which areexpressed exclusively on cancer cells, in that a pharmaceuticalcomposition according to the invention which comprises an mRNA whichcodes for such a cancer antigen is administered. By this means, thecancer antigen(s) is (are) expressed in the organism, as a result ofwhich an immune response which is directed effectively against thecancer cells is provoked.

In its use as a vaccine, the pharmaceutical composition according to theinvention is to be considered in particular for treatment of cancerdiseases (the mRNA preferably coding for a tumour-specific surfaceantigen (TSSA), e.g. for treatment of malignant melanoma, coloncarcinoma, lymphomas, sarcomas, small-cell pulmonary carcinoma,blastomas etc. Specific examples of tumour antigens are, inter alia,707-AP, AFP, ART-4, BAGE, β-catenine/m, Bcr-abl, CAMEL, CAP-1, CASP-8,CDC27/m, CDK4/m, CEA, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE,GnT-V, Gp100, HAGE, HER-2/neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M,HAST-2, hTERT (or hTRT), iCE, KIAA0205, LAGE, LDLR/FUT, MAGE,MART-1/melan-A, MC1R, myosine/m, MUC1, MUM-1, -2, -3, NA88-A, NY-ESO-1,p190 minor bcr-ab1, Pm1/RARα, PRAME, PSA, PSM, RAGE, RU1 or RU2, SAGE,SART-1 or SART-3, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2 and WT1.

According to a further preferred embodiment, the antigen(s) from atumour is or are a polyepitope of the antigen(s) from a tumour. A“polyepitope” of an antigen or several antigens is an amino acidsequence in which several or many regions of the antigen(s) whichinteract with the antigen-binding part of an antibody or with a T cellreceptor are represented. In this context, the polyepitope can becomplete and non-modified. However, according to the present inventionit can also be modified, in particular to optimize the antibody/antigenand T cell receptor/antigen interaction, respectively. A modificationcompared with the wild-type polyepitope can include e.g. a deletion,addition and/or substitution of one or more amino acid residues.Accordingly, in the mRNA of the present invention which codes for themodified polyepitope, one or more nucleotides is/are removed, addedand/or replaced, compared with the mRNA which codes for the wild-typepolyepitope.

In order to increase the stability of the (m)RNA contained in thepharmaceutical composition of the present invention, each (m)RNAcontained in the pharmaceutical composition preferably has one or moremodifications, in particular chemical modifications, which contributetowards increasing the half-life of the (m)RNA (one or more) in theorganism or improve the transfer of the (m)RNA (one or more) into thecell.

For example, in the sequences of eukaryotic mRNAs, there aredestabilizing sequence elements (DSE) to which signal proteins bind andregulate the enzymatic degradation of the mRNA in vivo. For furtherstabilization of the modified mRNA preferably contained in thepharmaceutical composition according to the invention, where appropriatein the region which codes for at least one antigen from a tumour one ormore modifications compared with the corresponding region of thewild-type mRNA are carried out, so that no destabilizing sequenceelements are present. According to the invention, it is of course alsopreferable, where appropriate, to eliminate from the mRNA DSEs presentin the untranslated regions (3′- and/or 5′-UTR).

Such destabilizing sequences are e.g. AU-rich sequences (“AURES”), whichoccur in 3′-UTR sections of numerous unstable mRNAs (Caput et al., Proc.Natl. Acad. Sci. USA 1986, 83: 1670 to 1674). The RNA moleculescontained in the pharmaceutical composition according to the inventionare therefore preferably modified compared with the wild-type mRNA suchthat they contain no such destabilizing sequences. This also applies tothose sequence motifs which are recognized by possible endonucleases,e.g. the sequence GAACAAG, which is contained in the 3′-UTR segment ofthe gene which codes for the transferrin receptor (Binder et al., EMBOJ. 1994, 13: 1969 to 1980). These sequence motifs are also preferablyeliminated in the modified mRNA of the pharmaceutical compositionaccording to the invention.

A skilled person in the art is familiar with various processes which aresuitable for substitution of codons in the modified mRNA according tothe invention. In the case of relatively short coding regions (whichcode for biologically active or antigenic peptides) e.g. the total mRNAcan be synthesized chemically using standard techniques.

Nevertheless, base substitutions are preferably introduced, using a DNAmatrix for the preparation of the modified mRNA with the aid oftechniques of the usual targeted mutagenesis; Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rded., Cold Spring Harbor, N. Y., 2001.

In this process, for the preparation of the mRNA, a corresponding DNAmolecule is therefore transcribed in vitro. This DNA matrix has asuitable promoter, e.g. a T7 or SP6 promoter, for the in vitrotranscription, which is followed by the desired nucleotide sequence forthe mRNA to be prepared and a termination signal for the in vitrotranscription. According to the invention, the DNA molecule which formsthe matrix of the RNA construct to be prepared is prepared byfermentative proliferation and subsequent isolation as part of a plasmidwhich can be replicated in bacteria. Plasmids which may be mentioned assuitable for the present invention are e.g. the plasmids pT7TS (GenBankAccess Number U26404; Lai et al., Development 1995, 121: 2349 to 2360;cf. also FIG. 8), pGEM® serie, e.g. pGEM®-1 (GenBank Access NumberX65300; from Promega) and pSP64 (GenBank Access Number X65327); cf. alsoMezei and Storts, Purification of PCR Products, in: Griffin and Griffin(ed.), PCR Technology: Current Innovation, CRC Press, Boca Raton, Fla.,2001.

Using short synthetic DNA oligonucleotides which contain shortsingle-stranded transitions at the cleavage sites formed or genesprepared by chemical synthesis, the desired nucleotide sequence can thusbe cloned into a suitable plasmid by molecular biology methods withwhich a skilled person in the art is familiar (cf. Maniatis et al., seeabove). The DNA molecule is then excised the plasmid, in which it can bepresent in one or multiple copy, by digestion with restrictionendonucleases.

The modified mRNA contained in the pharmaceutical composition accordingto the invention can moreover have a 5′-cap structure (a modifiedguanosine nucleotide). Examples of cap structures which may be mentionedare m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

According to a further preferred embodiment of the present invention,the modified mRNA contains a poly(A⁺) tail of at least about 25, inparticular at least about 30, preferably at least about 50 nucleotides,more preferably at least about 70 nucleotides, particularly preferablyat least about 100 nucleotides. However, the poly(A⁺) tail can alsocomprise 200 and more nucleotides.

For efficient translation of the mRNA, effective binding of theribosomes to the ribosome binding site (Kozak sequence: GCCGCCACCAUGG(SEQ ID NO: 2), AUG forms the start codon) is necessary. In thisrespect, it has been found that an increased A/U content around thissite renders possible a more efficient ribosome binding to the mRNA.

It is furthermore possible to insert one or more so-called IRES(“internal ribosomal entry site) into the mRNA. An IRES can thusfunction as the single ribosome binding site, but it can also serve toprovide an mRNA which codes several peptides or polypeptides which areto be translated by the ribosomes independently of one another(“multicistronic” or “polycistronic” mRNA). Examples of IRES sequenceswhich can be used according to the invention are those frompicornaviruses (e.g. FMDV), pestviruses (CFFV), polioviruses (PV),encephalomyocarditis viruses (ECMV), foot and mouth disease viruses(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),mouse leukoma virus (MLV), simian immunodeficiency viruses (SIV) orcricket paralysis viruses (CrPV).

According to a further preferred embodiment of the present invention,the mRNA has, in the 5′- and/or 3′-untranslated regions, stabilizingsequences which are capable of increasing the half-life of the mRNA inthe cytosol.

These stabilizing sequences can have a 100% sequence homology tonaturally occurring sequences which occur in viruses, bacteria andeukaryotes, but can also be partly or completely of synthetic nature.Examples of stabilizing sequences which can be used in the presentinvention and which may be mentioned are the untranslated sequences(UTR) of the β-globin gene, e.g. from Homo sapiens or Xenopus laevis.Another example of a stabilizing sequence has the general formula(C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC (SEQ ID NO: 1), which is containedin the 3′-UTR of the very stable mRNA which codes for α-globin,α-(I)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf. Holciket al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Suchstabilizing sequences can of course be used individually or incombination with one another and also in combination with otherstabilizing sequences known to a skilled person in the art.

For further stabilization of the mRNA, it is moreover preferred tocontain at least one analogue of naturally occurring nucleotides. Thisis based on the fact that the RNA-degrading enzymes occurring in thecells preferentially recognize naturally occurring nucleotides as asubstrate. The degradation of RNA can therefore be made difficult byinsertion of nucleotide analogues, whereby the effect on the translationefficiency on insertion of these analogues, in particular in the codingregion of the mRNA, can have a positive or negative effect on thetranslation efficiency.

In a list which is in no way conclusive, examples which may be mentionedof nucleotide analogues which can be used according to the invention arephosphoroamidates, phosphorothioates, peptide nucleotides,methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. Thepreparation of such analogues is known to a skilled person in the arte.g. from the U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S.Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707,U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No.5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat.Nos. 5,262,530 and 5,700,642. According to the invention, such analoguescan occur in untranslated and translated regions of the modified mRNA.

Furthermore, effective transfer of the preferably modified mRNA into thecells to be treated or the organism to be treated can be improved if themRNA is associated with a cationic or polycationic agent, in particulara corresponding peptide or protein, or bound thereto. The mRNA istherefore present in the pharmaceutical composition according to theinvention preferably in a form complexed or condensed with such anagent. In particular, the use of protamine as a polycationic, nucleicacid-binding protein is particularly effective in this context. The useof other cationic peptides or proteins, such as poly-L-lysine,poly-L-arginine or histones, is furthermore also possible. Thisprocedure for stabilizing the modified mRNA is described inEP-A-1083232, the disclosure content of which in this respect isincluded in its full scope in the present invention.

The mRNA modified according to the invention can moreover also contain,in addition to the peptide or polypeptide which is antigenic or activein gene therapy, at least one further functional section which e.g.codes for a cytokine which promotes the immune response, (monokine,lymphokine, interleukin or chemokine, such as IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN-α, IFN-γ, GM-CFS, LT-αor growth factors, such as hGH).

The pharmaceutical composition according to the invention can furthercomprise one or more adjuvants to increase the immunogenicity.“Adjuvant” here is to be understood as meaning any chemical orbiological compound which promotes a specific immune response. Variousmechanisms are possible in this respect, depending on the various typesof adjuvants used. For example, compounds which promote endocytosis ofthe modified mRNA contained in the pharmaceutical composition bydendritic cells (DC) form a first class of adjuvants which can be used.Other compounds which allow the maturation of the DC, e.g.lipopolysaccharides, TNF-α or CD40 ligand, are a further class ofsuitable adjuvants. Generally, any agent which influences the immunesystem of the nature of a “warning signal” (LPS, GP96, oligonucleotideswith the CpG motif) or cytokines, in particular GM-CSF, can be used asan adjuvant which allow an immune response against an antigen which iscoded by the modified mRNA to be increased and/or influenced in atargeted manner. In particular, the abovementioned cytokines arepreferred in this context. Further known adjuvants are aluminiumhydroxide, Freund's adjuvant and the abovementioned stabilizing cationicpeptides or polypeptides, such as protamine. Lipopeptides, such asPam3Cys, are also particularly suitable for use as adjuvants in thepharmaceutical composition of the present invention; c.f. Deres et al.,Nature 1989, 342: 561-564.

Further particularly suitable adjuvants are moreover (other) RNA or alsomRNA species, which can be added to the pharmaceutical composition ofthe present invention to increase the immunogenicity. Such adjuvant RNAis advantageously chemically modified for stabilization (“cismodification” or “cis stabilization”), for example by the abovementionednucleotide analogues, in particular phosphorothioate-modifiednucleotides, or by the above further measures for stabilization of RNA.A further advantageous possibility of stabilization is complexing orassociation (“trans association” or “trans modification” and “transstabilization”, respectively) with the abovementioned cationic orpolycationic agents, e.g. with protamine.

According to a further advantageous embodiment, the stability of the RNAmolecules contained in the pharmaceutical composition (mRNA, coding fora tumour antigen, and optionally adjuvant (m)RNA) is increased by one ormore RNase inhibitors. Preferred RNase inhibitors are peptides orproteins, in particular those from the placenta (e.g. from the humanplacenta) or pancreas. Such RNase inhibitors can also be in arecombinant form. A specific example of an RNase inhibitor is RNasin®,which is commercially obtainable, e.g. from Promega. Such RNaseinhibitors can be used generally for stabilizing RNA. A pharmaceuticalcomposition comprising at least one RNA, in particular mRNA, which codesfor at least one antigen, and at least one RNase inhibitor as definedabove, optionally in combination with a pharmaceutically acceptablesolvent, carrier and/or vehicle, is therefore also provided generallyaccording to the invention. Corresponding antigens in a general form andsolvents, carriers and vehicles are defined below. In respect ofpreferred tumour antigens, reference is made to the statements in thisrespect concerning the preferred pharmaceutical composition comprisingat least one mRNA which codes for at least one antigen from a tumour.

The pharmaceutical composition according to the invention preferablycomprises, in addition to the aqueous solvent and the mRNA, one or morefurther pharmaceutically acceptable carrier(s) and/or one or morefurther pharmaceutically acceptable vehicle(s). Corresponding routes forsuitable formulation and preparation of the pharmaceutical compositionaccording to the invention are disclosed in “Remington's PharmaceuticalSciences” (Mack Pub. Co., Easton, Pa., 1980), which is a constituent inits full content of the disclosure of the present invention. Possiblecarrier substances for parenteral administration are e.g., in additionto sterile water or sterile saline solutions as aqueous solvents, alsopolyalkylene glycols, hydrogenated naphthalene and, in particular,biocompatible lactide polymers, lactide/glycolide copolymers orpolyoxyethylene/polyoxypropylene copolymers. Compositions according tothe invention can comprise filler substances or substances such aslactose, mannitol, substances for covalent linking of polymers, such ase.g. polyethylene glycol, to inhibitors according to the invention,complexing with metal ions or inclusion of materials in or on particularpreparations of a polymer compound, such as e.g. polylactate,polyglycolic acid or hydrogel, or on liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte fragmentsor spheroplasts. The particular embodiments of the compositions arechosen according to the physical properties, for example in respect ofsolubility, stability, bioavailability or degradability. Controlled orconstant release of the active compound component according to theinvention in the composition includes formulations based on lipophilicdepots (e.g. fatty acids, waxes or oils).

Coatings of substances according to the invention or compositionscomprising such substances, that is to say coatings with polymers (e.g.polyoxamers or polyoxamines) are also disclosed in the context of thepresent invention. Substances or compositions according to the inventioncan furthermore have protective coatings, e.g. protease inhibitors orpermeability-increasing agents. Preferred aqueous carrier materials aree.g. water for injection (WFI) or water buffered with phosphate, citrateor acetate etc., whereby the pH typically being adjusted to 5.0 to 8.0,preferably 6.0 to 7.0. The aqueous solvent or the further carrier(s) orthe further vehicle(s) will additionally preferably comprise saltconstituents, e.g. sodium chloride, potassium chloride or othercomponents which render the solution e.g. isotonic. Aqueous solvents orthe further carrier(s) or the further vehicle(s) can furthermorecomprise, in addition to the abovementioned constituents, additionalcomponents, such as human serum albumin (HSA), Polysorbate 80, sugars oramino acids.

The method and mode of administration and the dosage of thepharmaceutical composition according to the invention depend on thedisease to be treated and the stage of advancement thereof, and also thebody weight, the age and the sex of the patient.

The concentration of the modified mRNA in such formulations cantherefore vary within a wide range from 1 μg to 100 mg/ml. Thepharmaceutical composition according to the invention is preferablyadministered to the patient parenterally, e.g. intravenously,intraarterially, subcutaneously or intramuscularly. It is also possibleto administer the pharmaceutical composition topically or orally. Thepharmaceutical composition according to the invention is preferablyadministered intradermally. A transdermal administration with the aid ofelectric currents or by osmotic forces is furthermore possible. Thepharmaceutical composition of the present invention can moreover beinjected locally into a tumour.

Thus, a method for treatment or a vaccination method for prevention ofcancer diseases or the abovementioned diseases which comprisesadministration of the pharmaceutical composition according to theinvention to a patient, in particular a human, is thus also providedaccording to the invention.

According to a preferred embodiment of the treatment or vaccinationmethod or in the use, defined above, of the mRNA according to theinvention which codes for at least one antigen from a tumour for thepreparation of a pharmaceutical composition for treatment and/orprevention of cancer diseases one or more cytokine(s) is administered tothe patient, in addition to the pharmaceutical composition according tothe invention.

A treatment or vaccination method comprising administration of at leastone RNA, preferably mRNA, which code(s) for at least one antigen from atumour (in accordance with the above definition) and is (are) optionallystabilized in accordance with the above statements, and at least onecytokine, e.g. one or more of the abovementioned cytokines, inparticular GM-CSF, to a patient, in particular a human, is thereforealso provided generally according to the invention. The method is usedin particular for treatment and/or prevention of corresponding cancerdiseases (e.g. the above cancer diseases). The present invention isaccordingly also directed generally to a pharmaceutical compositioncomprising at least one RNA, preferably mRNA, which code(s) for at leastone antigen from a tumour (according to the above definition) and is(are) optionally stabilized in accordance with the above statements, andat least one cytokine, e.g. one or more of the abovementioned cytokines,such as GM-CSF, preferably in combination with a pharmaceuticallyacceptable carrier and/or vehicle, e.g. an aqueous solvent, or one ormore of the carriers, solvents or vehicles defined above. The use ofcytokines, e.g. one or more of the abovementioned cytokines, inparticular GM-CSF, in combination with one or more RNA molecule(s) asdefined above, for treatment and/or prevention of cancer diseases (e.g.cancer diseases listed above) is thus also disclosed according to theinvention.

According to a further preferred embodiment of the present invention,the cytokine, e.g. GM-CSF, is administered simultaneously with or, whichis more preferable, before or after the pharmaceutical compositioncomprising the mRNA which codes for at least one antigen from a tumour(or is used for the preparation of a corresponding medicament forsimultaneous administration with or for administration before or afterthe abovementioned (m)RNA). The administration of the cytokine, inparticular GM-CSF, is very particularly preferably carried out shortlybefore (e.g. about 15 min or less, e.g. about 10 or about 5 min) or arelatively short time (e.g. about 5, 10, 15, 30, 45 or 60 min) after ora longer time (e.g. about 2, 6, 12, 24 or 36 h) after the administrationof the pharmaceutical composition defined above or generally after the(m)RNA of at least one which codes for at least one antigen from atumour.

The application of the cytokine, e.g. GM-CSF, can be carried out in thiscontext by the same route as the pharmaceutical composition according tothe invention or the at least one (m)RNA which codes for at least oneantigen from a tumour or in a manner separate from this. Suitableadministration routes and also the suitable formulation possibilities inrespect of the cytokine(s) can be found from the above statements inrespect of the pharmaceutical compositions according to the invention.In the case of a human patient, a GM-CSF dose of 100 micrograms/m² inparticular is advisable. The administration of the cytokine, e.g.GM-CSF, is particularly preferably carried out by an s.c. injection.

The pharmaceutical compositions of the present invention or the RNAwhich codes for an antigen from a tumour and where appropriate, inassociation therewith, the cytokine(s) are preferably administered inthe form of interval doses. For example, a dose of a pharmaceuticalcomposition according to the invention can be administered in relativelyshort intervals, e.g. daily, every second day, every third day etc., or,which is more preferable, in longer intervals, e.g. once weekly, once intwo weeks, once in three weeks, once a month etc. The intervals can alsobe changeable in this context, whereby it being necessary in particularto take into account the immunological parameters of the patient. Forexample, the administration of a pharmaceutical composition according tothe invention (and where appropriate, in association therewith, also theadministration of the cytokine(s)) can follow a treatment plan in whichthe interval is shorter, e.g. once in two weeks, at the start of thetreatment and then, depending on the course of treatment or theappropriately determined immunological parameters of the patient, theinterval is lengthened to e.g. once a month. A therapy plan tailor-madeto the particular individual can thus be applied according to thepatient, in particular his condition and his immunological parameters.

The present invention also provides a process for the preparation of thepharmaceutical composition defined above, comprising the steps:

-   (a) preparation of a cDNA library, or a part thereof, from tumour    tissue of a patient,-   (b) preparation of a matrix for in vitro transcription of RNA with    the aid of the cDNA library or a part thereof and-   (c) in vitro transcribing of the matrix.

The tumour tissue of the patient can be obtained e.g. by a simplebiopsy. However, it can also be provided by surgical removal oftumour-invaded tissue. The preparation of the cDNA library or a partthereof according to step (a) of the preparation process of the presentinvention can moreover be carried out after the corresponding tissue hasbeen deep-frozen for storage, preferably at temperatures below −70° C.For preparation of the cDNA library or a part thereof, isolation of thetotal RNA, e.g. from a tumour tissue biopsy, is first carried out.Processes for this are described e.g. in Maniatis et al., supra.Corresponding kits are furthermore commercially obtainable for this,e.g. from Roche AG (e.g. the product “High Pure RNA Isolation Kit”). Thecorresponding poly(A⁺) RNA is isolated from the total RNA in accordancewith processes known to a person skilled in the art (cf. e.g. Maniatiset al., supra). Appropriate kits are also commercially obtainable forthis. An example is the “High Pure RNA Tissue Kit” from Roche AG.Starting from the poly(A⁺) RNA obtained in this way, the cDNA library isthen prepared (in this context cf. also e.g. Maniatis et al., supra).For this step in the preparation of the cDNA library also, commerciallyobtainable kits are available to a person skilled in the art, e.g. the“SMART PCR cDNA Synthesis Kit” from Clontech Inc. The individualsub-steps from the poly(A⁺) RNA to the double-stranded cDNA is shownschematically in FIG. 11 by the example of the process in accordancewith the “SMART PCR cDNA Synthesis Kit” from Clontech Inc.

According to step (b) of the above preparation process, starting fromthe cDNA library (or a part thereof), a matrix is synthesized for the invitro transcription. According to the invention, this is effected inparticular by cloning the cDNA fragments obtained into a suitable RNAproduction vector. The suitable DNA matrix and the plasmids which arepreferred according to the invention are already mentioned above inconnection with the preparation of the mRNA for the pharmaceuticalcomposition according to the invention.

For in vitro transcription of the matrix prepared in step (b) accordingto the invention, these are first linearized with a correspondingrestriction enzyme, if they are present as circular plasmid (c)DNA.Preferably, the construct cleaved in this way is purified once more,e.g. by appropriate phenol/chloroform and/or chloroform/phenol/isoamylalcohol mixtures, before the actual in vitro transcription. By thismeans it is ensured in particular that the DNA matrix is in aprotein-free form. The enzymatic synthesis of the RNA is then carriedout starting from the purified matrix. This sub-step takes place in anappropriate reaction mixture comprising the linearized, protein-free DNAmatrix in a suitable buffer, to which a ribonuclease inhibitor ispreferably added, using a mixture of the required ribonucleotidetriphosphates (rATP, rCTP, rUTP and rGTP) and a sufficient amount of aRNA polymerase, e.g. T7 polymerase. The reaction mixture is present herein RNase-free water. Preferably, a CAP analogue is also added during theactual enzymatic synthesis of the RNA. After an incubation of anappropriately long period, e.g. 2 h, at 37° C., the DNA matrix isdegraded by addition of RNase-free DNase, incubation preferably beingcarried out again at 37° C.

Preferably, the RNA prepared in this way is precipitated by means ofammonium acetate/ethanol and, where appropriate, washed once or severaltimes with RNase-free ethanol. Finally, the RNA purified in this way isdried and, according to a preferred embodiment, is taken up inRNase-free water. The RNA prepared in this way can moreover be subjectedto several extractions with phenol/chloroform orphenol/chloroform/isoamyl alcohol.

According to a further preferred embodiment of the preparation processdefined above, only a part of a total cDNA library is obtained andconverted into corresponding mRNA molecules. According to the invention,a so-called subtraction library can therefore also be used as part ofthe total cDNA library in order to provide the mRNA molecules accordingto the invention. A preferred part of the cDNA library of the tumourtissue codes for the tumour-specific antigens. For certain tumours, thecorresponding antigens are known. According to a further preferredembodiment, the part of the cDNA library which codes for thetumour-specific antigens can first be defined (i.e. before step (a) ofthe process defined above). This is preferably effected by determiningthe sequences of the tumour-specific antigens by an alignment with acorresponding cDNA library from healthy tissue.

The alignment according to the invention comprises in particular acomparison of the expression pattern of the healthy tissue with that ofthe tumour tissue in question. Corresponding expression patterns can bedetermined at the nucleic acid level e.g. with the aid of suitablehybridization experiments. For this e.g. the corresponding (m)RNA orcDNA libraries of the tissue can in each case be separated in suitableagarose or polyacrylamide gels, transferred to membranes and hybridizedwith corresponding nucleic acid probes, preferably oligonucleotideprobes, which represent the particular genes (northern and southernblots, respectively). A comparison of the corresponding hybridizationsthus provides those genes which are expressed either exclusively by thetumour tissue or to a greater extent therein.

According to a further preferred embodiment, the hybridizationexperiments mentioned are carried out with the aid of a diagnosis bymicroarrays (one or more microarrays). A corresponding DNA microarraycomprises a defined arrangement, in particular in a small or very smallspace, of nucleic acid, in particular oligonucleotide, probes, eachprobe representing e.g. in each case a gene, the presence or absence ofwhich is to be investigated in the corresponding (m)RNA or cDNA library.In an appropriate microarrangement, hundreds, thousands and even tens tohundreds of thousands of genes can be represented in this way. Foranalysis of the expression pattern of the particular tissue, either thepoly(A⁺) RNA or, which is preferable, the corresponding cDNA is thenmarked with a suitable marker, in particular fluorescence markers areused for this purpose, and brought into contact with the microarrayunder suitable hybridization conditions. If a cDNA species binds to aprobe molecule present on the microarray, in particular anoligonucleotide probe molecule, a more or less pronounced fluorescencesignal, which can be measured with a suitable detection apparatus, e.g.an appropriately designed fluorescence spectrometer, is accordinglyobserved. The more the cDNA (or RNA) species is represented in thelibrary, the greater will be the signal, e.g. the fluorescence signal.The corresponding microarray hybridization experiment (or several ormany of these) is (are) carried out separately for the tumour tissue andthe healthy tissue. The genes expressed exclusively or to an increasedextent by the tumour tissue can therefore be concluded from thedifference between the signals read from the microarray experiments.Such DNA microarray analyses are described e.g. in Schena (2002),Microarray Analysis, ISBN 0-471-41443-3, John Wiley & Sons, Inc., NewYork, the disclosure content in this respect of this document beingincluded in its full scope in the present invention.

However, the establishing of tumour tissue-specific expression patternsis in no way limited to analyses at the nucleic acid level. Methodsknown from the prior art which serve for expression analysis at theprotein level are of course also familiar to a person skilled in theart. There may be mentioned here in particular techniques of 2D gelelectrophoresis and mass spectrometry, whereby these techniquesadvantageously also can be combined with protein biochips (i.e.,microarrays at the protein level, in which e.g. a protein extract fromhealthy or tumour tissue is brought into contact with antibodies and/orpeptides applied to the microarray substrate). With regard to the massspectroscopy methods, MALDI-TOF (“matrix assisted laserdesorption/ionization-time of flight”) methods are to be mentioned inthis respect. The techniques mentioned for protein chemistry analysis toobtain the expression pattern of tumour tissue in comparison withhealthy tissue are described e.g. in Rehm (2000) Der Experimentator:Proteinbiochemie/Proteomics [The Experimenter: ProteinBiochemistry/Proteomics], Spektrum Akademischer Verlag, Heidelberg, 3rded., to the disclosure content of which in this respect reference isexpressly made expressis verbis in the present invention. With regard toprotein microarrays, reference is moreover again made to the statementsin this respect in Schena (2002), supra.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures show:

FIG. 1 shows a graphical view of the results of a tumour vaccination,with RNA, of mice (rat Her-2/neu transgenic animals) which developmammary carcinomas spontaneously. The tumour multiplicity is plotted onthe y-axis against the age of the mice on the x-axis. Untreated mice(n=4), which served as a control, all had tumours at an age of 6 months.Three mice were injected with DNA which codes for Her-2/neu, one mousebeing tumour-free after 10 months. As a further negative control, 4 micereceived an antisense mRNA complementary to the mRNA for Her-2/neu.These mice also all had tumours after 6 months (not shown). In contrast,one of 4 mice which were injected with mRNA which codes for Her-2/neu(i.e., the sense strand) was tumour-free after 9 months.

FIG. 2 shows a graphical view of the results of experiments relating tobeta-galactosidase (beta-Gal)-specific CTL (cytotoxic T lymphocyte)activity by immunization with an mRNA which codes for beta-Gal, underthe influence of GM-CSF. BALB/c mice were immunized with 25 μg of mRNAwhich codes for beta-Gal by injection into the inner auricula. Thesplenocytes were stimulated with beta-Gal protein in vitro and the CTLactivity was determined 6 days after the in vitro stimulation using astandard ⁵¹Cr release test. The target cells were P815 (H₂ ^(d)) cellswhich were charged (▪) with the synthetic peptide TPHPARIGL, whichcorresponds to the H₂ ^(d) epitope of beta-Gal, or were not charged (▴).In each case three or two animals were treated per group. Animals whichwere injected i.d. in both auriculae with only injection buffer servedas a negative control. Animals which were injected i.d. in bothauriculae with 10 μg of a plasmid which codes for beta-Gal in PBS servedas a positive control (“DNA”). The test groups received RNA which codesfor beta-Gal by itself or in combination with GM-CSF, which was injected24 h (“GM-CSF t-1”), 2 h before the RNA injection (“GM-CSF t0”) or 24 hafter the RNA injection (“GM-CSF t+1”) into the same site (into theauriculae) or at another site (s.c. on the back). In each case threedifferent effector/target cell ratios (200, 44, 10) were tested.

FIGS. 3A-3B show further graphical views of the results of ELISAstandard tests specific for IFN-gamma (A) and IL-4 (B), which documentthe corresponding cytokine production of splenocytes which wererestimulated with beta-Gal protein in vitro. BALB/c mice were immunizedas already described above for FIG. 2. The splenocytes were stimulatedwith beta-Gal protein in vitro, the corresponding culture supernatantswere obtained and the IFN-gamma or IL-4 concentration was determinedusing an ELISA standard test.

FIGS. 4A-4B show further graphical views which demonstrate the antibodyresponse of mice immunized according to the invention. BALB/c mice wereimmunized as described for FIG. 2. Two weeks after the boost, blood wastaken and the blood serum was obtained therefrom. Beta-Gal-specific IgG1(A) and IgG2a antibodies (B) were determined with the aid of an ELISAtest. In each case the extinction (OD) at 405 nm which results from theconversion of the substrate ABTS in the ELISA test is shown on they-axis. The extinctions shown are the values from which thecorresponding values of mice treated with injection buffer aresubtracted.

FIG. 5 shows microscope sections, stained with X-Gal, of the auricula ofmice which have been injected i.d. into the auricula with mRNA whichcodes for beta-galactosidase. 12 hours after the injection of 25 μg RNAin HEPES-NaCl injection buffer, the ears were removed and sectionsstained with X-Gal were prepared. Blue cells indicate abeta-galactosidase activity. As can be seen from the two sections, onlyfew blue cells are present.

FIG. 6 shows a section, corresponding to FIG. 5, through an auricula ofa mouse which was injected into the auricula with mRNA which codes forbeta-galactosidase and was stabilized with protamine. The microscopesection stained with X-Gal show a few cells stained blue.

FIG. 7 shows two further sections through the auricula of mice, twoimages being produced per section in order to represent a larger area.In this case, mRNA which codes for beta-galactosidase, in a buffer, towhich 10 U RNasin, an enzymatic RNase inhibitor from the pancreas(obtainable from Roche or Promega) was added directly before theinjection, was injected into the auricula. Compared with the sections ofFIG. 5 and FIG. 6, significantly more blue-stained regions of cells withbeta-galactosidase activity are to be recognized.

FIG. 8 shows a schematical view of the plasmid pT7TS, which was used forthe in vitro transcription. Constructs according to the invention werecloned into the BglII and SpeI sites, the relative position of which toone another is shown. The region shaded in black contains the 5′untranslated region of the beta-globin gene from Xenopus laevis, whilethe region shaded in grey represents a corresponding 3′ untranslatedregion of the beta-globin gene from X. laevis (SEQ ID NO: 4). Therelative position of the T7 promoter (SEQ ID NO: 3), the PstI site usedfor sequencing, the poly(A⁺) tail (A₃₀C₃₀) and, with an arrow, thetranscription direction are furthermore indicated.

FIG. 9 shows in a flow chart, by way of example, the course of an RNAvaccination therapy according to the invention with assistingadministration of GM-CSF. The mRNA molecules which code for one or moretumour antigens (MUC1, Her-2/neu, tilomerase, MAGE-1) or a mRNA whichcodes for a control antigen (influenza matrix protein (IMP), a viralantigen) are administered i.d. to the patient on days 0, 14, 28 and 42.In addition, one day after the RNA inoculation the patient is injecteds.c. with GM-CFS (Leucomax® (100 μg/m²) from Novartis/Essex Pharma).When the course is stable or there is an objective tumour response(complete remission (CR) or partial remission (PR)), the patientsreceive the vaccinations s.c. once a month. After the fourth injection(day 49), the response of the tumour is evaluated radiologically, bylaboratory chemistry or sonographically, and the immunological phenomenainduced by the therapy are evaluated. From day 70, the immunizationtherapy is continued at intervals of 4 weeks. On day 0, 14, 28, 42 and49, blood samples are taken for determination of appropriate laboratoryparameters, the differential blood count (Diff-BB), FACS analysis andcytokines. Restaging of the patient takes place from day 49 and whereappropriate every further 4 to 8 weeks.

FIG. 10 shows a flow chart of the construction of autologous, stabilizedRNA according to the preparation process of the present invention.Tumour tissue is first obtained, e.g. by biopsy. The total RNA isextracted from this. A cDNA library is constructed with the aid of thepoly(A⁺) RNA obtained from the RNA extraction. Starting from this, afterpreparation of a corresponding DNA matrix, the autologous, stabilizedRNA is obtained by means of in vitro transcription.

FIG. 11 shows a reaction scheme of the steps for preparation of a cDNAlibrary, starting from poly(A⁺) RNA, for the SMART PCR cDNA SynthesisKit from Clontech Inc. by way of example.

FIG. 12 shows a photograph of an agarose gel which shows the typicalsize fractionation of a cDNA library compiled from human placentatissue. A length marker with fragments of the length shown on the leftis plotted in track M. The “DS cDNA” track contains the cDNA library.Those fragments which correspond to the expected size fraction (about200 bp to 4,000 bp) are used for the in vitro transcription.

FIG. 13 shows by way of example a treatment plan for the tumour therapyaccording to the invention by injection of a tumour mRNA library, herein combination with GM-CSF, for patients with malignant melanoma.Autologous, stabilized RNA prepared from the patient's own tumour tissueis used for this. This amplified autologous tumour RNA is administeredto the patient i.d. on days 0, 14, 28 and 42. In addition, one day afterthe RNA injection the patient is injected s.c. with GM-CSF (Leucomax®100 μg/m² Novartis/Essex Pharma). Two weeks after the fourth injection(day 56), the response of the tumour is evaluated by a staging analysis(inter alia sonography, thorax X-ray, CT etc.) and by assessment of theimmunological parameters induced by the therapy. When the course of thedisease is stable or there is an objective tumour response (CR or PR),the patient receives in each case a further vaccination every fourweeks. Further restaging analyses are carried out on day 126 and then atintervals of 12 weeks.

FIG. 14 shows once more schematically of the general course of a therapywith the pharmaceutical composition according to the invention withautologous, amplified tumour RNA, i.e. the RNA contained in thepharmaceutical composition represents a cDNA library of the tumourtissue. A sample of the tumour tissue is first obtained, e.g. via abiopsy. The total and then the poly(A⁺) RNA are prepared from the tissueby appropriate extractions. Starting from the poly(A⁺) RNA, a cDNAlibrary is constructed and is cloned into a vector suitable forsubsequent in vitro transcription. An RNA vaccine is then obtained by invitro transcription, and is injected into the patient from whom thetumour tissue has been taken to combat the tumour.

EXAMPLES

The following embodiment examples explain the present invention in moredetail, without limiting it.

Example 1 Tumour Vaccination with RNA in an Animal Model

Materials and Methods

Capped mRNA which codes for a shortened version of the Her-2/neu proteinof the rat (“ECD-TM-neu-rat”, containing the extracellular domain andthe transmembrane region, but not the cytoplasmic region) was prepared,using the “SP6 mMessagemMachine” (Ambion), with the aid of a plasmidwhich substantially corresponded to the structure shown in FIG. 8, butcontained an SP6 promoter instead of the T7 promoter and in which theECD-TM-neu-rat construct was inserted after the SP6 RNA polymerasepromoter. The mRNA prepared was dissolved in injection buffer (150 mMNaCl, 10 mM HEPES) at a concentration of 0.8 mg/l and the solution wasmixed with protamine sulfate (Sigma) (1 mg protamine per 1 mg RNA). 50μl of this solution were injected into the auriculae (in each case 25 μlper ear) of mice. Eight injections were performed, in each case one atthe age of 6, 8, 13, 15, 20, 22, 27 and 29 weeks. Mice to whichcorresponding injections with injection buffer, with plasmid DNA whichcodes for ECD-TM-neu rat or with an antisense mRNA corresponding to themRNA according to the invention were administered served as controls.

Results

Female BalB-neu T mice (BalB/c mice which express the oncogene Her-2/neuof the rat; cf. Rovero et al. (2000) J. Immunol. 165(9):5133-5142) whichdevelop mammary carcinomas spontaneously were immunized with RNA whichcodes for a shortened version of the Her-2/neu protein(“ECD-TM-neu-rat”, containing the extracellular domains and thetransmembrane region, but not the cytoplasmic region). Four mice treatedwith injection buffer served as a negative control. A further group ofthree mice was injected with DNA which codes for the shortenedHer-2/neu. Four mice received the mRNA which codes, according to theinvention, for the tumour antigen Her-2/neu (shortened version ofECD-TM, see above). Four mice which were injected with the correspondingantisense RNA served as a further control group. As shown in FIG. 1, inthe animals of the untreated control group a tumour multiplicity of onaverage 10 was observed after 26 weeks, whereby all animals havingpalpable breast tumours at the age of about 20 weeks. In contrast, inthe case of immunization with the mRNA which codes for ECD-TM-neu-rat, asignificant slowing down of the formation of carcinomas is to beobserved, in particular a tumour multiplicity of 10 is achieved only atthe age of 30 weeks. Furthermore, the size of the tumours is alsoreduced (not shown). Of the 4 mice treated with the mRNA according tothe invention, one was still tumour-free after 9 months. That group ofmice which had been injected with the antisense mRNA all showed tumoursat the age of 6 months. The comparison group of mice injected withplasmid DNA which codes for the shortened version of Her-2/neu alsoshowed a carcinoma formation which was slowed down compared with theuntreated control group (cf. also in respect of corresponding plasmidDNA experiments on intramuscular injection: Di Carlo et al. (2001) Clin.Cancer Res. 7 (3rd supplement): 830s-837s), but the formation ofcarcinomas up to the 27th week was not slowed down to the same extent asin the case of immunization with mRNA according to the invention whichcodes for the shortened version of Her-2/neu. Furthermore, in the caseof immunization with DNA, the abovementioned disadvantages, inparticular the risk of integration of the DNA into the genome, theformation of anti-DNA antibodies etc., are to be taken into account.

Example 2 Influence of GM-CSF on RNA Vaccination

Materials and Methods

Mice

BALB-c AnNCrlBR (H-2^(d)) mice (female) 6-10 weeks old were obtainedfrom Charles River (Sulzfeld, Germany).

Plasmids and Preparation of RNA

The ORF (LacZ) which codes for beta-galactosidase, flanked by 5′- and3′-untranslated sequences from the beta-globin gene of X. Laevis, wasinto the plasmid pT7TS (P.A. Creek, Austin, Tex., USA), in order toprepare the plasmid pT7TS-kozak-5′ beta gl-lacZ-3′ beta gl-A30C30 (cf.Hoerr et al. (2000) Eur. J. Immonol. 30: 1-7). A schematical view of thegeneral structure of the plasmid pT7TS with the flanking 5′ and 3′untranslated sequences from the beta-globin gene of X. Laevis is shownin FIG. 8.

The plasmid prepared in this way was linearized with PstI andtranscribed in vitro using the m-MessagemMachineT7 Kit (Ambion, Austin,Tex. USA). The RNA prepared in this way was purified by means of LiClprecipitation, phenol/chloroform extraction and ammonium acetateprecipitation. Finally, the purified RNA was resuspended in injectionbuffer (150 mM NaCl, 10 mM HEPES) in a concentration of 0.5 mg/ml.

Media and Cell Culture

P815 and P13.1 cells were cultured in RPMI 1640 (Bio-Whittaker,Verviers, Belgium), supplemented with 10% heat-inactivated foetal calfserum (FCS) (PAN systems, Germany), 2 mM L-glutamine, 100 U/mlpenicillin and 100 mg/ml streptomycin.

CTL cultures were kept in RPMI 1640 medium, supplemented with 10% FCS, 2mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.05 μMbeta-mercaptoethanol, 50 mg/ml gentamycin, MEM non-essential amino acids(100×) and 1 mM sodium pyruvate. The CTL were restimulated for one weekwith 1 mg/ml beta-galactosidase protein (Sigma, Taufkirchen, Germany).On day 4, 4 ml of culture supernatant were carefully pipetted off andreplaced by fresh medium containing 10 U/ml rIL-2 (final concentration).

Immunization

3 BALB/c mice per group were anesthetized with 20 mg pentobarbital i.p.per mouse. The mice were then injected i.d. in both auriculae with 25 mgof mRNA which codes for beta-galactosidase (beta-Gal) in injectionbuffer (150 mM NaCl, 10 mM HEPES). In some cases, granulocyte macrophagecolony-stimulating factor (GM-CSF) was additionally injected into thesame site or into an injection site away from this (into the auricula ors.c. into the back) 24 h or 2 h before or 24 h after the RNA injection.As a positive control, animals were injected i.d. in both auriculae within each case 10 mg of a DNA plasmid which codes for beta-gal in PBS. Agroup of animals to which only injection buffer was administered i.d.into both auriculae served as a negative control. Two weeks after thefirst injection, a boost injection was performed in each case in thesame manner as the first injection. Two weeks after the boost injection,blood was taken, the mice were sacrificed and the spleen was removed.

⁵¹Cr Release Test

Splenocytes obtained from the spleen were stimulated with beta-galprotein in vitro and the CTL activity was determined after 6 days usinga 6-hours ⁵¹Cr standard test as described in Rammensee et al. (1989)Immunogenetics 30: 296-302. Summarized briefly, target cells were markedwith ⁵¹Cr and charged with the peptide TPHPARIGL for 20 min at roomtemperature. After co-incubation of effector and target cells (at ineach case three different ratios of effector:target cells: 200, 44 and10) in circular plates with 96 wells for 6 h, 50 ml of 200 ml of culturesupernatant were pipetted into a Luma scintillation plate (Packard) with96 wells and, after drying, the radioactivity was measured with ascintillation counter (1405 Microbeta Plus). The percentage specificrelease was determined from the amount of ⁵¹Cr released into the medium(A) minus the spontaneous release (B) divided by the total release (C)(using Triton X-100) minus the spontaneous release (B): Percent specificlysis=100 (A−B)/(C−B).

Cytokine ELISA

After 4 days of restimulation with beta-gal protein, the supernatant ofthe splenocyte culture was pipetted off and stored at −50° C. untilused. 100 ml anti-mouse-anti-IFN-gamma or -IL-4 scavenger antibodies(Becton Dickenson, Heidelberg, Germany) were pipetted out overnight at4° C. on MaxiSorb plates (Nalge Nunc International, Nalge, Denmark) at aconcentration of 1 mg/ml in coating buffer (0.02% NaN₃, 15 mM Na₂CO₃, 15mM NaHCO₃, pH 9.6). After washing three times with washing buffer (0.05%Tween 20 in PBS), the plates were saturated with 200 ml of blockingbuffer (0.05% Tween 20, 1% BSA in PBS) for 2 h at 37° C. After washingthree times with washing buffer, 100 ml of the cell culture supernatantswere incubated for 5 h at 37° C. The plates were then washed four timeswith washing buffer, 100 ml of biotinylated anti-mouse-anti-IFN-gamma or-IL-4 detection antibodies (Becton Dickenson, Heidelberg, Germany) perwell at a concentration of 0.5 mg/ml in blocking buffer were pipettedand incubation was carried out for 1 h at room temperature. Afterwashing three times with washing buffer, 100 ml of a 1/1,000 dilution ofstreptavidin-HRP (BD Biosciences, Heidelberg, Germany) were added intoeach well. After 30 min at room temperature, the plates were washedthree times with washing buffer and twice with bidistilled water.Thereafter, 100 ml of the ABTS substrate were added into each well.After 15-30 min at room temperature, the extinction at 405 nm wasmeasured with a Sunrise ELISA reader (Tecan, Crailsheim, Germany).

Antibody ELISA

Two weeks after the boost injection, blood was taken from the mice viathe orbital vein and blood serum was prepared. 100 ml of beta-galprotein at a concentration of 100 mg/ml in coating buffer (0.05 MTris-HCl, 0.15 M NaCl, 5 mM CaCl₂, pH 7.5) were pipetted out for 2 h at37° C. on to MaxiSorb plates (Nalge Nunc International, Nalge, Denmark).The plates were then washed three times with 200 ml of washing buffer(0.05 M Tris-HCl, 0.15 M NaCl, 0.01 M EDTA, 0.1% Tween 20, 1% BSA, pH7.4) and saturated with protein with 200 ml of washing buffer overnightat 4° C. The plates were washed three times with washing buffer andblood sera were added in a dilution of 1/10, 1/30 or 1/90 in washingbuffer. After 1 h at 37° C., the plates were washed three times withwashing buffer and 100 ml of 1/1,000 dilutions of goat anti-mouse IgG1or IgG2a antibodies (Caltag, Burlington, Calif., USA) were added. After1 h at room temperature, the wells were washed three times with washingbuffer and 100 ml of ABTS substrate per well were added. After 15-30 minat room temperature, the extinction at 405 nm was measured with aSunrise ELISA reader (Tecan, Crailsheim, Germany).

Results and Discussion

It was confirmed that direct injection of RNA which codes forbeta-galactosidase into the auricula of mice induces ananti-beta-galactosidase immune response, substantially of the Th2 type.Production of anti-beta-galactosidase immunoglobulins of the IG1 type(FIG. 3A) and secretion of IL-4 (FIG. 3B) was found in splenocytes,stimulated with beta-galactosidase, from mice which had been injectedwith the RNA which codes for beta-galactosidase. To increase theefficiency of the RNA vaccine, the cytokine GM-CSF was additionallyadministered. This cytokine increases the efficiency of some DNAvaccines. It was furthermore found that the time of the GM-CSF injectioninfluences the type of the immune response, compared with DNA injection(Kusakabe (2000) J. Immunol. 164: 3102-3111). It was found according tothe invention that GM-CSF can enhance the immune response brought aboutby an RNA vaccination. The injection of GM-CSF one day before theinjection of RNA shows scarcely any influence on the strength or thetype of the immune response. In contrast, injection of GM-CSF 2 hoursbefore injection of the RNA enhances the immune response (cf. the IL-4release in FIG. 3B in the 2 mice injected with GM-CSF at time T=0), butdoes not influence the Th2 polarity. On the other hand, if GM-CSF isinjected one day after the RNA vaccine into the same site or into a siteaway from this (not shown), not only is the immune response enhancedoverall (cf. the antibody response according to FIG. 3), the immuneresponse is polarized to the Th1 type (cf. the IFN-gamma production bysplenocytes stimulated with beta-gal protein according to FIG. 3A, theproduction of IgG2a antibodies against beta-Gal according to FIG. 3B andthe production of activated CTL according to FIG. 1). The injection ofGM-CSF some minutes or some hours after the RNA injection should resultin the same effect (enhancement and polarization) on the immuneresponse.

Example 3 Effect of an RNase Inhibitor on mRNA Expression In Vivo

Naked or protamine-associated or -complexed mRNA which codes forbeta-galactosidase (prepared as described in example 2) was injectedinto the auricula of mice in an amount of 25 mg of RNA in injectionbuffer (150 mM NaCl, 10 mM HEPES). Further mice were injected with themRNA which codes for beta-galactosidase, together with 10 U of the RNaseinhibitor RNasin (an enzymatic RNase inhibitor extracted from thepancrease, obtainable from Roche or Promega). The RNase inhibitor wasmixed with the RNA solution directly before the injection. After 12hours, the ears were in each case removed from the mice. Thin microscopesections of the auriculae were prepared and were stained with X-gal.Injection of naked or protamine-associated mRNA leads to a detectablebeta-galactosidase activity in a few cells in the corresponding thinsections (blue cells in FIGS. 5 and 6). Some cells have thus taken upthe exogenous RNA here and translated it into the protein. When the mRNAwhich codes for beta-galactosidase was in the form protected with theRNase inhibitor RNasin, very many more blue cells were observed than inthe case of the naked or protamine-associated RNA (FIG. 7). Since RNasininhibits RNases, the half-life of the injected mRNA molecules in vivo isprolonged, where the environment (interstitial tissue) is contaminatedwith RNases. Such a stabilization of the RNA leads to an increaseduptake by the surrounding cells and therefore to an increased expressionof the protein coded by the exogenous RNA. This phenomenon can thereforealso be utilized for an enhanced immune response to an antigen coded bythe mRNA injected.

Example 4 RNA Vaccination of Patients with Malignant Diseases

Introduction

Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides (8-9amino acids) which are expressed bound to MHC class 1 glycoproteins onthe cell surface (1). These peptides are fragments of intracellularprotein molecules. However, there are indications that antigens taken upexogenously by macropinocytosis or phagocytosis can lead to the CD8⁺ Tcell-mediated immune response. The proteins are cleaved into proteosomesand the peptides formed by this means are transported out of the cytosolinto the lumen of the endoplasmic reticulum and bound to MHC class Imolecules.

The proteins processed in this way are transported as peptide/MHC classI complex to the cell surface and presented to the CTL. This processtakes place in every cell and in this way makes it possible for theimmune system to monitor accurately each individual cell for thepresence of proteins which are foreign to the body or modified orembryonic, regardless of whether they originate from intracellularpathogenic germs, oncogenes or dysregulated genes. By this means,cytotoxic lymphocytes are capable of recognizing and lysing infected andneoplastic cells, respectively (2, 3).

In recent years various tumour-associated antigens (TAA) and peptideswhich are recognized by CTL and therefore lead to lysis of tumour cellshave been successfully isolated (21-27). These TAA are capable ofstimulating T cells and inducing antigen-specific CTL, if they areexpressed as a complex of HLA molecule and peptide on antigen-presentingcells (APC).

In numerous studies carried out mainly on patients with malignantmelanoma, it has been possible to demonstrate that malignant cells losethe expression of TAA as the tumour disease proceeds. Similarcircumstances are also observed with vaccinations with individual tumourantigens. Under vaccination therapies, selection of tumour cells mayalso occur, which renders possible an escape from the immune system anda progression of the disease in spite of therapy. The use of severaldifferent tumour antigens as envisaged in the treatment plan accordingto the invention of the present example should prevent selection oftumour cells and escape of the malignant cells from the immune systemdue to loss of antigens.

A method with which DC can be transfected with RNA from a plasmid whichcodes for a tumour antigen has recently been developed (Nair et al.,1998, Nair et al., 2000). Transfection of DC with RNA for CEA ortelomerase led to induction of antigen-specific CTL. This processrenders it possible to induce CTL and T helper cells against severalepitopes on various HLA molecules from a tumour antigen. A furtheradvantage of this strategy is the fact that neither the characterizationof the tumour antigens or epitopes used nor definition of the HLAhaplotype of the patient is a prerequisite. By a polyvalent vaccine ofthis type, the probability of the occurrence of so-called clonal “tumourescape” phenomena could be reduced significantly. Furthermore, Tcell-mediated immune responses against antigens processed and presentedby the natural route and with possibly a higher immune dominance couldbe induced by this approach. By additional participation of MHC classII-restricted epitopes, the induced tumour-specific immune responsecould be enhanced and maintained for longer.

A treatment scheme according to the invention for tumour vaccination ofpatients with advanced malignant diseases (mammary, ovarian, colorectal,pancreatic and renal cell carcinomas) is provided by way of example. Inthis, RNA which has been prepared from plasmids which code for MUC1,Her-2/neu, telomerase and MAGE-1 tumour antigens and influenza matrixprotein (IMP) (positive control) is administered intradermally topatients with the abovementioned malignant diseases. A CTL induction invivo is thereby rendered possible, in order to prevent the progressionof the disease or to effect the regression thereof in this way. Thetumour antigens mentioned are expressed on the malignant cells ofmammary, ovarian, colorectal, pancreatic and renal cell carcinomas.

According to the treatment plan (cf. the following statements in thisrespect and FIG. 9), the RNA species prepared in the laboratory whichcode for CEA, MUC1, Her-2/neu, telomerase, Mage-1 and IMP areadministered to the patient i.d., initially 4× on days 0, 14, 28 and 42.In addition, GM-CSF (Leucomax®, 100 μg/m², Novartis/Essex Pharma) isadministered s.c. to the patient in each case one day after the RNAinoculation.

The treatment according to the invention is an immunisation approachwhich requires only minimal interventions on the patient (injection).Therapy is conducted ambulant and is suitable for many tumour patients,without the limitation to particular HLA types or defined T cellepitopes. Furthermore, polyclonal CD4⁺-T helpers and also CD8⁺-CTL canbe induced by this therapy.

Treatment Plan

The RNAs for several tumour antigens (MUC1, Her-2/neu, telomerase,MAGE-1) and for a control antigen, influenza matrix protein (IMP, aviral antigen) are administered i.d. to the patient on days 0, 14, 28and 42. In addition, the patients receive GM-CSF (Leucomax® (100 μg/m²)Novartis/Essex Pharma) s.c. in each case one day after the RNAinoculation. When the course of the disease is stable or there is anobjective tumour response (complete remission (CR) or partial remission(PR)), where appropriate the patients receive the vaccinations s.c. oncea month. After the fourth injection (day 49), the response of the tumouris evaluated radiologically, by laboratory chemistry and/orsonographically, and the immunological phenomena induced by the therapyare evaluated.

From day 70, the immunization therapy is continued at intervals of 4weeks.

On days 0, 14, 28, 42 and 49, in each case blood samples are taken forlaboratory parameters, Diff-BB, FACS analysis and cytokines (50 ml intotal). Restaging of the patients takes place from day 49 and whereappropriate every further 4 to 8 weeks.

The treatment plan is shown schematically in FIG. 9.

-   Laboratory: clotting, electrolytes, LDH, β2-M, CK, liver enzymes,    bilirubin, creatinine, uric acid, total protein, CRP, tumour markers    (Ca 12-5, Ca 15-3, CEA, Ca 19-9): 15 ml of blood.-   Diff-BB: differential blood count with smear (5 ml of EDTA blood).-   Cytokines: 10 ml of serum-   FACS: 10 ml of heparin blood.-   ELIspot: 20 ml of heparin blood.-   Multitest: analysis of the DTH reaction.-   DTH: (“delayed type hypersensitivity”, delayed T cell-mediated    reaction) analysis of the reaction to intradermally administered    RNA. In addition a skin biopsy should be performed in the event of a    positive DTH reaction (local anaesthesia is not necessary for this).    Preparation of RNA from Plasmids

For production of a vaccine based on mRNA, only precursors which arechemically synthesized and purified from bacteria are required. This ispreferably effected in a specially equipped RNA production unit. This isin a sealed-off room which is declared an RNase-free zone, i.e. workwith RNase (e.g. purification of plasmids) must not be carried out.Contamination with naturally occurring RNases is also constantlychecked. This room is fitted out with new apparatuses (4° C. and −20° C.refrigerators, heating block, sterile bench, centrifuges, pipettes)which have never been used for biological or clinical work. This RNAproduction unit is used exclusively for enzymatic production (in vitrotranscription) of mRNA (without bacterial, viral or cell culture work).The end product comprises a sterile RNA solution in HEPES/NaCl buffer.Quality analyses are carried out on a formaldehyde-agarose gel. Inaddition, the RNA concentration and the content of proteins aredetermined photometrically (OD₃₂₀<0.1; ratio of OD₂₆₀/OD₂₈₀>1.8 in pureRNA). Possible contamination by LPS is analysed in the LAL test. All RNAsamples are subjected to sterile filtration before administration.

Plasmid Constructs

The chosen genes (CEA, mucin1, Her-2/neu, telomerase, Mage-A1 andinfluenza matrix) are amplified via a PCR using a heat-stablehigh-performance enzyme (pfu, Stratagene). The genes originate fromtumour cDNA (mucin1, Her-2/neu, telomerase), or they have been clonedinto bacterial vectors (influenza matrix and MAGE-A1). The PCR fragmentsare cleaved with restriction enzymes (mucin1: BglII-SpeI; Her-2/neu:HinDIIIblunt-SpeI; telomerase: BglII-SpeI; MAGE-A1: BamHI-SpeI;influenza matrix protein: BglII-SpeI) and cloned into the T7TS-Plasmid(cf. FIG. 8) via the BglII and SpeI restriction sites. Plasmids of highpurity are obtained via the Endo-free Maxipreparation Kit (Qiagen,Hilden, Germany). The sequence of the vector is controlled via adouble-strand sequencing from the T7 promoter up to the PstI site anddocumented. Plasmids with a correct inserted gene sequence withoutmutations are used for the in vitro transcription. (Control via thepublished sequences: Accession Numbers: M11730 for Her-2/neu, NM_002456for MUC1, NM_003219 for telomerase TERT, V01099 for influenza matrix andM77481 for MAGE-A1).

In Vitro Transcription

Production of Linear, Protein Free DNA

500 μg of each plasmid are linearized in a volume of 0.8 ml viadigestion with the restriction enzyme PstI in a 2 ml Eppendorf reactionvessel. This cleaved construct is transferred into the RNA productionunit. 1 ml of a mixture of phenol/chloroform/isoamyl alcohol is added tothe linearized DNA. The reaction vessel is vortexed for 2 minutes andcentrifuged at 15,000 rpm for 3 minutes. The aqueous phase is removedand mixed with 0.7 ml 2-propanol in a 2 ml reaction vessel. This vesselis centrifuged at 15,000 rpm for 15 minutes, the supernatant isdiscarded and 1 ml 75% ethanol is added. The reaction vessel iscentrifuged at 15,000 rpm for 10 minutes and the ethanol is removed. Thevessel is centrifuged for a further 2 minutes and the residues of theethanol are removed with a microliter pipette tip. The DNA pellet isthen dissolved in 1 μg/ml in RNase-free water.

Enzymatic Synthesis of the RNA

The following reaction mixture is prepared in a 50 ml Falcon tube: 100μg linearized protein-free DNA, 1 ml 5× buffer (200 mM Tris-HCl (pH7.9), 30 mM MgCl₂, 10 mM spermidine, 50 mM NaCl, 50 mM DTT), 200 μlribonuclease (RNase) inhibitor (recombinant, 5,000 U), 1 ml rNTP mix (ineach case 10 mM ATP, CTP, UTP; 2 mM GTP), 1 ml CAP analogue (8 mM), 150μl T7 polymerase (3,000 U) and 2.55 ml RNase-free water. The totalvolume is 5 ml. The mixture is incubated at 37° C. for 2 hours in aheating block. Thereafter, 100 U of RNase-free DNase are added and themixture is incubated again at 37° C. for 30 minutes. The DNA matrix isenzymatically degraded by this procedure.

Description and Origin of the Individual Components

T7 polymerase: purified from an E. coli strain which contains a plasmidwith the gene for the polymerase. This RNA polymerase uses as thesubstrate only promoter sequences of the T7 phage; Fermentas.

NTPs: synthesized chemically and purified via HPLC. Purity more than96%; Fermentas.

CAP analogue: synthesized chemically and purified via HPLC. Purity morethan 90%; Institute of Organic Chemistry of the University of Tübingen.

RNase inhibitor: RNasin, for injection, prepared recombinantly (E.coli); Promega.

DNase: Pulmozym® (“dornase alfa”); Roche

Purification

The RNA treated with DNase is mixed with 20 ml of a solution of 3.3 ml 5M NH₄OAc plus 16.65 ml of ethanol. The mixture is incubated at −20° C.for 1 hour and centrifuged at 4,000 rpm for 1 hour. The supernatant isremoved and the pellet is washed with 5 ml of 75% RNase-free ethanol.The vessel is centrifuged again at 4,000 rpm for 15 minutes and thesupernatant is removed. The vessel is centrifuged again under theprevious conditions and the ethanol which remains is removed with amicroliter pipette tip. The reaction vessel is opened and the pellet isdried under a sterile bench in the sterile environment.

1 ml of RNase-free water is added to the dried RNA. The pellet isincubated at 4° C. for at least 4 hours. 2 μl of the aqueous solutionare subjected to a quantitative analysis (determination of the UVabsorption at 260 nm). 2 ml of a phenol/chloroform/isoamyl alcoholsolution are added to 1 ml of aqueous RNA solution. The mixture isvortexed for 2 minutes and centrifuged at 4,000 rpm for 2 minutes. Theaqueous phase is removed with a microliter pipette and transferred intoa new reaction vessel. 4 ml of a solution of 0.66 ml 5 M NH₄OAc plus3.33 ml ethanol are added. The mixture is incubated at −20° C. for 1hour and centrifuged at 4,000 rpm for 1 hour. The supernatant is removedand the pellet is washed with 75% RNase-free ethanol. The vessel iscentrifuged again at 4,000 rpm for 15 minutes and the supernatant isremoved. The vessel is centrifuged again under the previous conditionsand the ethanol which remains is removed with a microliter pipette tip.The reaction vessel is opened and the pellet is dried under a sterilebench in the sterile environment.

The RNA is dissolved in RNase-free water and adjusted to a concentrationof 10 mg/ml. It is incubated for 12 hours at 4° C. A final concentrationof 2 mg/ml is achieved by addition of injection buffer (150 mM NaCl, 10mN HEPES). The end product is preferably subjected to sterile filtrationunder GMP conditions before use.

Application of the RNA

Each patient receives at two different sites an intradermal (i.d.)injection of in each case 150 μl of the injection solution in which ineach case 100 μg of antigen-coding mRNA (CEA, Her-2/neu, MAGE-A1, mucin1, telomerase, influenza matrix protein) are present in solution.

After the primary immunization, a booster immunization is carried outevery 14 days, for the inoculations then to be repeated at a monthlyinterval. In each case one day after the RNA injection, GM-CSF(Leucomax®, Sandoz/Essex Pharma) is administered subcutaneously (s.c.)to the patient.

If a clinical response is present or the disease is stabilized, thistherapy is continued at monthly intervals.

Further Immunological Investigations In Vitro (Optional)

Flow cytometry analyses of PBMC for quantification of CTL precursors;

⁵¹Cr release tests;

Soluble receptor and cytokine levels in the serum;

DTH reaction (skin reaction to intradermally injected RNA, “delayed typehypersensitivity”, T lymphocyte-mediated reaction); and

Skin biopsy samples from the injection site for histological analysisfor T cell infiltration (pathology).

Parameters for Evaluation of the Efficacy

To be able to answer the question of the efficacy of this immunotherapy,the induction of tumour-specific T cells and a measurable tumourremission is used. Parameters are T cell reactions measured in vitro andin vivo and changes in the size of bidimensionally recordable tumourmanifestation or laboratory chemistry parameters of the course of thedisease.

Objective remission is defined as the best response in the form of acomplete or partial remission, corresponding to the criteria listedbelow. The remission rate is calculated from the ratio of the number ofpatients with objective remission and the total number of evaluablepatients.

A change in the immune status, determined by immunotyping of peripheralmononuclear cells, an increase in the antigen-specific CTL precursorfrequency in the peripheral blood and the induction of a persistenttumour-specific T cell activity are assessed as the immunologicalresponse to the therapy. For this purpose, in vitro induction culturesare established for activation of tumour-specific CTL.

Remission Criteria (acc. to UICC)

-   Complete remission (CR): Complete regression of all measurable    tumour manifestation, documented by 2 control investigations at    least 4 weeks apart.-   Partial remission (PR): Decrease in size of the total area    dimensions (product of two tumour diameters or linear measurement of    one-dimensionally measurable lesions of all tumour findings by 50%    for at least 4 weeks). No new occurrence of tumour manifestations or    progression of a tumour finding.-   “No Change” (NC): Decrease of all the measurable tumour    manifestations by less than 50% or increase in a tumour finding.-   Progression (PD): Increase in size of the tumour parameters in at    least one focus or new occurrence of a tumour manifestation.

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Example 5 Vaccination with Autologous, Amplified Tumour RNA in Patientswith Malignant Melanoma

Introduction

The incidence of malignant melanoma has increased sharply worldwide inrecent years. If the melanoma disease is already in the metastased stageat the time of diagnosis, there is currently no therapy which has apositive influence on the further course of the disease with sufficientcertainty.

Vaccination therapies carried out to date using dendritic cells are verylabour-, cost- and time-intensive because of the complicated culturingof the cells (GMP conditions). Furthermore, the studies have hithertoconcentrated predominantly on known tumour-associated antigens (TAA),such as, for example, melan-A or tyrosinase.

A number of various immunological phenomena, such as, inter alia, theoccurrence of spontaneous tumour regressions or spontaneous involutionof metastases, have made the melanoma the prior candidate for testingimmunotherapy investigations (Parkinson et al., 1992). In addition toexperiments on non-specific stimulation of the immune system by means ofinterleukin-2, mistletoe extracts, BCG and interferons, which have sofar not led to decisive breakthroughs in the therapy of advanced tumourdiseases, the strategy of induction of various highly specific cytotoxicT lymphocytes (CTL) has been pursued in particular in recent years.These CTL are capable of recognizing and killing autologous melanomacells (Boon et al., 1994; Houghton, 1994). Studies of this process haveshown that the CTL recognize defined peptides in combination with MHCclass I molecules. The presentation of peptides by antigen-presentingcells (APC) is the physiological route to generation of specific immuneresponses by lymphocytes (Rammensee, 1993). Dendritic cells have provedto be potent antigen-presenting cells which lead to an induction of theimmune response by two routes: The first is the direct presentation ofpeptides towards CD8⁺-T lymphocytes and activation thereof (Schuler &Steinmann, 1985; Inaba et al., 1987; Romani et al., 1989), and thesecond is the generation of a protective immune response, which ismediated by CD4⁺ helper lymphocytes, and requires a presentation ofpeptides via MHC class II molecules (Grabbe et al., 1991, 1992, 1995).

By means of peptide analysis, it was therefore possible to identify inthis way various tumour-associated antigens (TAA) which are specific forthe melanoma and, after presentation in combination with the MHCmolecule and recognition by the CTL, lead to cytolysis of the tumourcells (Schadendorf et al., 1997, p. 21-27).

The use of autologous, dendritic cells was tested in the context of apilot study on melanoma patients in respect of its potential to inducecytotoxic T lymphocytes effectively, rapidly and reliably. In thisstudy, 16 melanoma patients in stage IV who had already been pretreatedby chemotherapy were vaccinated with peptide-charged dendritic cells.The response rates were above 30% (5/16 patients) (Nestle et al., 1998).In a further independent study it was possible to demonstrate an evenhigher response rate of more than 50% (6/11 patients) after immunizationof melanoma patients who had already been pretreated by chemotherapywith MAGE-3A1-charged dendritic cells (Thurner et al., 1999). Asignificant expansion of MAGE-A3-specific CD8⁺-T cells was also observedin 8/11 patients. A regression of the metastases took place in somecases after the DC vaccination. This was accompanied by a CD8⁺-T cellinfiltration. This showed that the T cells induced were active in vivo.A disadvantage of this strategy is the high outlay on costs and thelaboratory (in particular GMP conditions). Large amounts of blood fromthe patient are required for the time-intensive generation of the DC. Inthe preparation of the peptides, on the one hand only knowntumour-associated antigens can be used, and on the other hand variouspeptides are necessary, depending on the HLA haplotype.

A further development of this approach is vaccination withRNA-transfected DC (Nair et al., 1998, Nair et al., 2000). In themeantime, numerous studies demonstrate that DC from mice and humanswhich have been transfected with mRNA can induce an efficient CTLresponse in vitro and in vivo and can lead to a significant reduction inmetastases (Boczkowski et al., 1996, 2000; Ashley et al., 1997; Nair etal., 1998, 2000; Heiser et al., 2001; Mitchell and Nair, 2000; Koido etal., 2000; Schmitt et al., 2001). A great advantage in the use of RNAcompared with peptides is that the most diverse peptides can beprocessed and presented from one mRNA which codes for a TAA. By apolyvalent vaccine of this type, the probability of the occurrence ofso-called clonal “tumour escape” phenomena can be reduced significantly.Furthermore, T cell-mediated immune responses against antigens processedand presented by the natural route and with potentially a higher immunedominance can be induced by this system. By additional participation ofMHC class II-restricted epitopes, the tumour-specific immune responseinduced can be intensified and maintained for longer. Nevertheless, thisprocess also can be carried out only with a high outlay on thelaboratory (GMP conditions) because of the necessary culturing of theautologous DCs.

In the present strategy according to the invention, vaccination iscarried out with the RNA expression profile present in the autologoustumour of the patient. The specific tumour profile of the patient isthereby taken into account, unknown TAAs also being included in theinoculation. Expensive culture of the DCs is omitted, since RNAs (nottransfected DCs) are used in the vaccination.

A vaccination therapy using amplified autologous tumour RNA on patientswith metastased malignant melanoma, in particular stage III/IV, istherefore provided according to the invention. Tumour-specific cytotoxicT cells are induced in vivo by the vaccination, in order thus to achievea clinico-therapeutic effect (tumour response). This is an immunisationsystem which requires only minimal interventions on the patient(injection). Therapy can be conducted ambulant and is suitable for manytumour patients, without the limitation to particular HLA types ordefined T cell epitopes. Furthermore, polyclonal CD4⁺-T helpers and alsoCD8⁺-CTL can be induced by this therapy. From the point of view of thestrategy, it is decisive also that hitherto unknown TAAs are taken intoaccount in the vaccination protocol, and the exclusive use of autologousmaterial is particularly advantageous.

Treatment Plan

The amplified autologous tumour RNA is administered to the patient i.d.on days 0, 14, 28 and 42. In addition, the patients receive GM-CSF(Leucomax® 100 μg/m², Novartis/Essex) s.c. in each case one day afterthe RNA inoculation. Each patient receives, at two different sites, ani.d. injection of in each case 150 μl of the injection solution, inwhich in each case 100 μg of autologous tumour RNA is dissolved.

2 weeks after the fourth injection (day 56), where appropriate theresponse of the tumour is evaluated by a staging analysis (inter aliasonography, thorax X-ray, CT etc.; in this context see the statementsbelow) and by assessment of the immunological parameters induced by thetherapy.

When the course of the disease is stable or there is an objective tumourresponse (CR or PR), the patients receive the vaccinations every fourweeks. Further restaging analyses can be envisaged e.g. on day 126 andthen at an interval of 12 weeks.

A diagram of the treatment plan is shown in FIG. 13.

Preparation of Autologous Tumour RNA

The aim is the preparation of autologous poly(A⁺) RNA. For this,poly(A⁺) RNA is isolated from the patient's own tumour tissue. This RNAisolated is very unstable per se and its amount is limited. The geneticinformation is therefore transcribed into a considerably more stablecDNA library and thus conserved. Starting from the patient's own cDNAlibrary, stabilized autologous RNA can be prepared for the entiretreatment period. The procedure according to the invention is shownschematically in FIG. 10.

Isolation of RNA

A process of Roche AG is used to isolate total RNA from a tumour tissuebiopsy. The High Pure RNA Isolation Kit (order number 1828665) isemployed here in accordance with the manufacturer's instructions.Poly(A⁺) RNA is isolated from the total RNA via a further process ofRoche AG with the High Pure RNA Tissue Kit (order number 2033674).

Preparation of a cDNA Library

The cDNA library is constructed with the “SMART PCR cDNA Synthesis Kit”(Clontech Inc., USA; order number PT3041-1) in accordance with themanufacturer's instructions.

In this procedure, the single-stranded poly(A⁺) RNA is subjected toreverse transcription via a specific primer. Via a poly-C overhang atthe 3′-end of the newly synthesized DNA, a further primer can hybridize,via which the construct can be amplified by a PCR. The double-strandedcDNA fragments are now ready for cloning into a suitable RNA productionvector (e.g. pT7TS; cf. FIG. 8).

The process for the preparation of the cDNA library from the poly(A⁺)RNA with the aid of the above kit is shown schematically in FIG. 11.

Plasmid Constructs

The cDNA PCR fragments are cleaved with the restriction enzymes NotI andSpeI and cloned into the corresponding restriction sites of the pT7TSvector by a procedure analogous to that described in example 4. Plasmidsof high purity are obtained via the Endo-free Maxipreparation Kit(Qiagen, Hilden, Germany). Plasmids with a cloned-in gene sequence whichcorresponds to the expected size fractionation (200 bp-4,000 bp) of thecDNA library are used for the in vitro transcription. An example of aseparation of a representative cDNA library in an agarose gel is shownin FIG. 12.

In Vitro Transcription and RNA Administration

The in vitro transcription and the administration of the RNA are carriedout as described in the above example 4.

Investigations During the Treatment

Before each inoculation (on the day of the inoculation):

Physical examination (including RR, fever);

Blood sample for routine laboratory values

-   1. Blood count, differential blood count: 3 ml-   2. Electrolytes, LDH, CK, liver enzymes, bilirubin, creatinine, uric    acid, total protein, CRP: 5 ml-   3. Blood sedimentation: 2 ml; and-   at repeat inoculations additionally: Inspection of the injection    sites.

On day 1 after each inoculation:

Physical examination (including RR, fever); and

Inspection of the injection sites.

In staging analyses on day 56 and 126 after the first inoculation, thenevery 12 weeks, additionally:

Extended routine blood sample:

1. Tumour marker S 100 (7 ml)

2. Clotting values (3 ml);

Blood sample for immune monitoring (30 ml);

General well-being (ECOG score);

Imaging methods (thorax X-ray, sonography, skeleton scintigram, CTabdomen, pelvis, thorax, skull); and

ECG (“EKG”).

Further Immunological Investigations In Vitro

Where appropriate, the relative incidence of antigen-specific CTLprecursor cells in the peripheral blood of the patient in the course oftime of the vaccination therapy is measured.

On the one hand CTL precursor cells which are directed against antigensexpressed to a particular degree by melanoma cells (tyrosinase, MAGE-3,melan-A, GP100) are quantified here with FACS analyses (tetramerstaining). On the other hand ELIspot analyses are carried out, thesebeing designed such that CTL precursor cells which are directedspecifically against hitherto unknown antigens are additionallyrecorded. For this, autologous dendritic cells cultured from theperipheral blood of the patient are incubated with the same RNA withwhich the inoculation has also been carried out. These then serve asstimulator cells in the ELIspot analysis. The measurement thus recordsthe total vaccine spectrum. For these analyses, blood samples of 30 mlin total (20 ml ELIspot, 10 ml FACS analysis) can be envisaged for theimmune monitoring in the context of the staging analyses andadditionally on days 0, 14, 28 and 42, as well as a single withdrawal of100 ml on day 70 for culture of the DC.

Furthermore, skin biopsy samples from the injection site can be obtainedfor histological analysis in respect of a T cell infiltration.

Parameters for Evaluation of the Efficacy

The efficacy of the therapy according to the invention is evaluated withthe aid of the parameters described above in example 4.

REFERENCES

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The invention claimed is:
 1. A method of stimulating an antitumor immuneresponse in a subject comprising administering an effective amount of acell-free composition comprising mRNA encoding an MAGE antigen to asubject in need thereof, thereby stimulating a T-cell mediated cytotoxicanticancer immune response in the subject.
 2. The method of claim 1,wherein the subject has a cancer.
 3. The method of claim 2, wherein thecancer is a lung or skin cancer.
 4. The method of claim 1, wherein thecomposition comprises mRNA encoding at least 2, 3, 4 or 5 differenttumor antigens.
 5. The method of claim 1, wherein the method furthercomprises administering at least 2, 3, 4 or 5 different cell-freecompositions comprising mRNA encoding different tumor antigens to thesubject.
 6. The method of claim 1, wherein the mRNA is complexed with atleast one cationic or polycationic agent.
 7. The method of claim 6,wherein the cationic or polycationic agent is chosen from the groupconsisting of protamine, poly-L-lysine, poly-L-arginine and histones. 8.The method of claim 7, wherein the mRNA is complexed with protamine. 9.The method of claim 1, further comprising administering one or moreadjuvant(s) to the subject.
 10. The method of claim 9, wherein theadjuvant is chosen from the group consisting of lipopolysaccharide,TNF-α, CD40 ligand, GP96, oligonucleotides with a CpG motif, aluminumhydroxide, Freund's adjuvant, a lipopeptide and a cytokine.
 11. Themethod of claim 10, wherein the cytokine is GM-CSF.
 12. The method ofclaim 1, wherein the mRNA encoding the antigen has a different nucleicacid sequence compared with the wild-type mRNA encoding the antigen. 13.The method of claim 1, wherein the mRNA comprises a 5′ cap structure, atleast one IRES and/or a poly(A⁺) tail of at least 25 nucleotides. 14.The method of claim 13, wherein the mRNA comprises a 5′ cap structureand a poly(A⁺) tail of at least 25 nucleotides.
 15. The method of claim1, wherein the mRNA comprises at least one 5′-stabilizing sequenceand/or at least one 3′-stabilizing sequence.
 16. The method of claim 15,wherein the 5′- and/or the 3′-stabilizing sequence(s) is/are chosen fromthe group consisting of untranslated sequences (UTR) of the β-globingene and a stabilizing sequence of the general formula(C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC.
 17. The method of claim 1, whereinthe mRNA comprises at least one analog of naturally occurring nucleotideselected from the group consisting of phosphorothioates,phosphoroamidates, peptide nucleotides, methylphosphates,7-deazaguanosine, 5-methylcytosine and inosine.
 18. The method of claim1, wherein the cell-free composition comprising mRNA is administered byinjection of an aqueous solution comprising the mRNA.
 19. The method ofclaim 1, wherein the cell-free composition comprising mRNA isadministered intradermally.
 20. The method of claim 1, wherein thecell-free composition comprising mRNA is administered two or more times.